The theory of non-specific effects (NSEs) of vaccines proposes that vaccines in high-mortality populations have substantial impacts on mortality rates that are not explained by the prevention of targeted diseases, nor conversely by typical expected adverse reactions to the vaccines, and that these NSEs are generally more pronounced in females. The existence of these effects, and any implications for the development of vaccines and the design of vaccination programs to enhance safety, remain controversial. The long-term goal of the proposed research is to contribute to the overall understanding of vaccine safety in high-mortality populations, through the study of NSEs in a novel animal model system, free-roaming domestic dogs. The overall objectives in this application are (i) to confirm preliminary data that adjuvanted, non-live animal rabies vaccine (ARV) substantially increases all-cause mortality rates in female but not male animals in this system, (ii) to determine if non-adjuvanted, non-live human rabies vaccine (HRV) also has an effect on all-cause mortality in dogs that is modified by sex, and (iii) to quantify the extent to which the sex-differential effect of vaccine type on mortality is mediated by the nature of the immune response elicited. The central hypothesis is that adjuvanted ARV in dogs has a detrimental effect on female survival by modulating the immune response to infectious and/or parasitic diseases to which females are normally less susceptible than males, and that non-adjuvanted HRV will not have this same detrimental effect. The rationale for this project is that controlled studies of NSEs at low risk of bias are needed to achieve scientific consensus on the topic, and that while such studies may be challenging to conduct in children in high-mortality settings, they are ethically and logistically feasible in the proposed animal model system. The central hypothesis will be tested by pursuing two specific aims: 1) Determine if ARV and HRV have an effect on survival in dogs that is modified by sex, and 2) Determine to what extent the effect of vaccine type on survival is mediated by a shift towards a Th2-type cytokine response shortly after vaccination. These aims will be achieved in a randomized, three-arm, owner-blind, sham- controlled animal trial. Dogs will be randomly allocated to one of 3 treatment groups (ARV, HRV or sham- injection). The primary outcome will be death due to any cause. For the second aim, statistical mediation analysis will be used to quantify what proportion of the effect of vaccine type on survival is mediated through a shift towards a Th2-type cytokine response following vaccination. The research proposed in this application is innovative because it focuses on establishment of an animal model for randomized controlled trials at low risk of bias in high-mortality populations, which will allow for integration of studies of mechanistic links between vaccine exposures, immunological changes and non-specific survival outcomes. The research is significant because it is expected to provide evidence of sex-differential non-specific effects of non-live vaccines in a high- mortality population. Ultimately, such knowledge may offer new opportunities to enhance vaccine safety.